Method for preparing an aqueous solution enriched in both EG-III &amp; xylanase using a low molecular weight alcohol and an organic salt

ABSTRACT

A method for preparing an aqueous solution enriched in xylanase only, EG III only and both EG III and xylanase from an aqueous mixture containing cellulase proteins, xylanase and EG III is disclosed. The methods involve adding an amount of a low molecular weight alcohol and an organic salt to an aqueous mixture containing cellulase proteins under conditions wherein substantially all of the cellulase proteins other than EG III and xylanase are precipitated out of the aqueous mixture. The methods can then involve adding an inorganic salt to the supernate produced in the previous step so as to form a second precipitate and a second supernate and then finally collecting either the second precipitate from the second supernate to obtain a precipitate enriched in xylanase or the second supernate from the second precipitate to obtain a supernate enriched in EG III.

This is a continuation of application Ser. No. 07/862,641 filed on Apr.3, 1992, now U.S. Pat. No. 5,320,960.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to methods for producing an aqueoussolution containing a substantially pure EG III cellulase component. Inparticular, the methods of the present invention are directed, in part,to the removal of cellulase proteins, except the EG III cellulasecomponent, from an aqueous mixture of cellulase proteins containing EGIII by the addition of a low molecular weight alcohol to the aqueousmixture in the presence of an organic salt. In a preferred embodiment,the pH of the aqueous mixture is adjusted to at least about pH=7 beforethe addition of the alcohol. In another preferred embodiment, aninorganic salt is added to the EG III-rich supernate to precipitate theremaining contaminating proteins resulting in a substantially pure EGIII composition. The methods of the present invention are also directedin part to the enrichment of xylanase from an aqueous solutioncontaining xylanase.

2. State of the Art

Cellulases are known in the art as enzymes that hydrolyze cellulose(β-1,4-glucan linkages) thereby resulting in the formation of glucose,cellobiose, cellooligosaccharides, and the like. While cellulases areproduced (expressed) in fungi, bacteria and the like, cellulase producedby certain fungi and, in particular by the fungus class Trichoderma spp.(especially Trichoderma reesei), have been given the most attentionbecause a complete cellulase system capable of degrading crystallineforms of cellulose is readily produced in large quantities viafermentation procedures.

In regard to the above, Schulein, "Methods in Enzymology", 160, 25,pages 234 et seq. (1988), disclose that complete fungal cellulasesystems comprise several different enzyme classifications includingthose identified as exo-cellobiohydrolases (EC 3.2.1.91) ("CBH"),endoglucanases (EC 3.2.1.4) ("EG"), and β-glucosidases (EC 3.2.1.21)("BG"). The fungal cellulase classifications of CBH, EG and BG can befurther expanded to include multiple components within eachclassification. For example, multiple CBHs and EGs have been isolatedfrom a variety of fungal sources.

The complete cellulase system comprising CBH, EG and BG components isrequired to efficiently convert crystalline cellulose to glucose.Isolated components are far less effective, if at all, in hydrolyzingcrystalline cellulose. Moreover, a synergistic relationship is observedbetween the cellulase components particularly if they are of differentclassifications.

On the other hand, cellulases and components thereof, used eithersingularly or in combination, are also known in the art to be useful indetergent compositions. For example, endoglucanase components of fungalcellulases have been used for the purposes of enhancing the cleaningability of detergent compositions, for use as a softening agent, and foruse in improving the feel of cotton fabrics, and the like. However,there is a problem with using the EG I and EG II components derived fromTrichoderma spp. and especially Trichoderma reesei in detergentcompositions. Specifically, such components have their maximal activityat acidic pHs whereas most laundry detergent compositions are formulatedfor use at neutral or alkaline (pH>7 to about 10) conditions. While itis disclosed in U.S. Ser. No. 07/668,640 that the use of one or moreacidic endoglucanase components of Trichoderma reesei in detergentcompositions will provide improvements in softening, colorretention/restoration and feel to cotton-containing fabrics even whentreated under alkaline conditions, it is disclosed in U.S. Ser. No.07/707,647 that the EG III component of Trichoderma spp. provides forsuperior and unexpected advantages in detergent compositions as comparedto the EG I and EG II components of Trichoderma reesei.

Specifically, the EG III cellulase component has been found to possesssignificant enzymatic activity under alkaline conditions and isparticularly suited for use in laundry conditions where a neutral oralkaline detergent wash medium is employed.

In addition to its use in laundry detergents, the substantially pure EGIII cellulase component described herein can additionally be used in apre-washing step in the appropriate solution at an intermediate pH wheresufficient activity exists to provide desired improvements in colorretention/restoration, softening and feel as disclosed in U.S. Ser. No.07/707,647 filed May 30, 1991 and incorporated herein by reference.

Also, it is contemplated that the substantially pure EG III cellulasecomponent described herein can be used in home use as a stand alonecomposition suitable for restoring color to faded fabrics (see, forexample, U.S. Pat. No. 4,738,682, which is incorporated herein byreference in its entirety) as well as used in a spot-remover.

Additionally, it is further contemplated that the high activity underneutral to alkaline conditions of the EG III cellulase component wouldbe beneficial in textile processes for treating cotton-containingfabrics (see U.S. Ser. Nos. 07/677,385 and 07/678,865 which areincorporated herein by reference in their entirety) as well as in silageand/or composting processes.

In contrast to the above, this invention is directed to efficientprocesses for the separation and purification of the EG III cellulasecomponent from aqueous enzyme mixtures, particularly from a completecellulase composition and particularly for commercial scale productionof the EG III cellulase component.

SUMMARY OF THE INVENTION

Specifically, the present invention is directed to a method forproducing an aqueous solution containing substantially pure EG IIIcellulase component from an aqueous mixture containing cellulaseproteins including EG III cellulase component. Accordingly, in one ofits method aspects, the present invention is directed to a method forselectively removing substantially all of the cellulase proteins, otherthan the EG III component, from the aqueous mixture containing cellulaseproteins including EG III cellulase which method comprises the additionto the aqueous mixture of an effective amount of a low molecular weightalcohol in the presence of an organic salt under conditions whereinsubstantially all of the other cellulase proteins are precipitated fromsolution, and removing the precipitate. In a preferred embodiment, thepH of the aqueous mixture is adjusted to at least about pH=7 before theaddition of alcohol. In another preferred embodiment of this invention,an inorganic salt is added to the EG III-rich supernate and allremaining cellulase proteins, other than EG III, are precipitated.

The methods of the present invention are also directed in part, to theisolation of an aqueous solution containing substantially pure xylanasefrom Trichoderma spp.

The aqueous mixture can be a filtered whole cell extract or, morepreferably, a whole cellulase composition from a wild-type Trichodermaspp. strain, a genetically modified Trichoderma spp. strain, or anyother aqueous mixture, compatible with the methods of this invention andcontaining cellulase proteins including EG III.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the RBB-CMC activity profile over a pH range at 40°C. for an EG enriched fungal cellulase composition derived from a strainof Trichoderma reesei transformed so as to be incapable of expressingCBH I and CBH II; as well as the activity profile of an enriched EG IIIcellulase composition derived from Trichoderma reesei over a pH range at40° C.

FIG. 2 is an isoelectric focusing gel which, in Lane 1 displays theproteins expressed by a wild type Trichoderma reesei, in Lane 2 displaysthe proteins expressed by a strain of Trichoderma reesei transformed soas to be incapable of expressing EG I and EG II components; and in Lane3 displays the proteins found in substantially pure EG III cellulase.The right hand margin of this figure is marked so as to identify thebands attributable to CBH I, CBH II, EG I, EG II EG III and xylanase.

FIG. 3 is the amino acid sequence obtained from two fragments of EG III.

FIG. 4 is an SDS-PAGE gel which in Lane 11 displays the proteinsexpressed by a strain of Trichoderma reesei transformed so as to beincapable of expressing EG I and EG II components; in Lane 2 displaysthe proteins found in substantially pure EG III cellulase obtained bythe method of Part A in Example I; and in Lane 12 displays pure EG IIIobtained by the method of Example 2.

FIG. 5 is an outline of the construction of pΔCBHIpyr4.

FIG. 6 illustrates deletion of the T. reesei gene by integration of thelarger EcoRI fragment from pΔCBHIpyr4 at the cbh1 locus on one of the T.reesei chromosomes.

FIG. 7 is an autoradiograph of DNA from T. reesei strain GC69transformed with EcoRI digested pΔCBHIpyr4 after Southern blot analysisusing a ³² P-labelled pΔCBHIpyr4 as the probe. The sizes of molecularweight markers are shown in kilobase pairs to the left of the Figure.

FIG. 8 is an autoradiograph of DNA from a T. reesei strain GC69transformed with EcoRI digested pΔCBHIpyr4 using a ³² P labelledpIntCBHI as the probe. The sizes of molecular weight markers are shownin kilobase pairs to the left of the Figure.

FIG. 9 is an isoelectric focusing gel displaying the proteins secretedby the wild type and by transformed strains of T. reesei. Specifically,in FIG. 5, Lane A of the isoelectric focusing gel employs partiallypurified CBHI from T. reesei; Lane B employs a wild type T. reesei: LaneC employs protein from a T. reesei strain with the cbh1 gene deleted;and Lane D employs protein from a T. reesei strain with the cbh1 andcbh2 genes deleted. In FIG. 9, the right hand side of the figure ismarked to indicate the location of the single proteins found in one ormore of the secreted proteins. Specifically, BG refers to theβ-glucosidase E1 refers to endoglucanase I, E2 refers to endoglucanaseII, E3 refers to endoglucanase III, C1 refers to exo-cellobiohydrolase Iand C2 refers to exo-cellobiohydrolase II.

FIG. 10A is a representation of the T. reesei cbh2 locus, cloned as a4.1 kb EcoRI fragment on genomic DNA and FIG. 10B is a representation ofthe cbh2gene deletion vector pPΔCBHII.

FIG. 11 is an autoradiograph of DNA from T. reesei strain P37PΔCBHIPyr26transformed with EcoRI digested pPΔCBHII after Southern blot analysisusing a ³² P labelled pPΔCBHII as the probe. The sizes of molecularweight markers are shown in kilobase pairs to the left of the Figure.

FIG. 12 is an outline of the construction of pΔEGIpyrG-3.

FIG. 13 illustrates deletion of the egl1 gene by integration of theHindIII fragment from pΔEGIpyrG-3 at the egl1 locus on one of the T.reesei chromosomes.

FIG. 14 is an outline of the construction of pAΔEGII-1.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention generally relates to methods forproducing substantially pure EG III cellulase component whether in anaqueous solution or as a recovered protein.

However, prior to discussing this invention in further detail, thefollowing terms will first be defined:

1. Definitions

As used herein, the following terms have the following meanings:

"EG III cellulase" refers to the endoglucanase component derived fromTrichoderma spp. or any microorganism producing a protein equivalent toEG III produced by Trichoderma spp. characterized by a pH optimum ofabout 5.5 to 6.0, an isoelectric point (pI) of from about 7.2 to 8.0 anda molecular weight of about 23 to 28 Kdaltons. Preferably, EG IIIcellulase is derived from either Trichoderma reesei or from Trichodermaviride. EG III cellulase derived from Trichoderma reesei has a pHoptimum of about 5.5 to 6.0, an isoelectric point (pI) of about 7.4 anda molecular weight of about 25 to 28 Kdaltons. EG III cellulase derivedfrom Trichoderma viride has a pH optimum of about 5.5, an isoelectricpoint (pI) of about 7.7 and a molecular weight of about 23.5 Kdaltons.Additionally, it is contemplated that the amino acid sequence of the EGIII cellulase may be altered. Alteration of the active sites on thisenzyme may lead to a variety of different changes such as different pHoptima, different temperature optima or altered affinities for thesubstrate.

Because of its high pI, the EG III component is found in a region of anisoelectric focusing gel where high pI xylanases and other high pIcomponents expressed by Trichoderma spp. are generally found. In fact,it has been hypothesized that the band identified as EG III in FIG. 2was a degradation product of either EG I or II. However, gel isoelectricfocusing gels of EG I and EG II deleted cellulase (prepared in themanner of U.S. Ser. Nos. 07/770,049 and 07/668,640 and as described inExamples 18 and 19 hereinbelow) demonstrated that this band was notattributable to a degradation product of either EG I or II. (See alsoFIG. 2).

It is noted that EG II has been previously referred to by thenomenclature "EG III"by some authors but current nomenclature uses theterm "EG II". In any event, the EG II protein is substantially differentfrom the EG III protein in its molecular weight, pI, and pH optimum asevidenced by Table I of Example 2 presented below.

"Substantially pure EG III component" refers to a an aqueous solution orcomposition of cellulase proteins containing at least 50 weight percent,more preferably at least 70 weight percent and most preferably at least90 weight percent of EG III cellulase component based on the totalweight of the cellulase proteins in the composition.

"Substantially free of other cellulase proteins" refers to a compositionin which at least 50 weight 10 percent, more preferably 60 weightpercent and most preferably at least 90 weight percent of the cellulaseproteins, other than EG III, have been removed from the original aqueousmixture of cellulase proteins.

"Enriched in xylanase" refers to an aqueous solution or compositioncontaining an increase in xylanase concentration by the processes ofthis invention by at least a factor of 4, more preferably by at least afactor of 10.

"Cellulase proteins" refers to cellulase proteins which contain any andall exo-cellobiohydrolase (CBH) proteins, endoglucanase (EG) proteinsand β-glucosidase (BG) proteins derived from fungal sources ormicroorganisms genetically modified so as to incorporate and express allor a part of the cellulase genes obtained from a fungal source.

Collectively, all of such proteins (i.e. CBH, EG and BG proteins) arereferred to as "cellulase proteins". Certainly, cellulase proteins donot include other proteins expressed by Trichoderma spp. includingxylanases, proteases, amylases, etc.

"Endoglucanase (EG) components" refer to the EG components ofTrichoderma spp. including the EG I, EG II and/or EG III components ofTrichoderma reesei. The endoglucanase components of Trichoderma spp.(e.g., the EG I, EG II, EG III components of Trichoderma reesei, and thelike) either alone or in combination, impart improved feel, improvedappearance, softening, color enhancement, and/or a stone washedappearance to cotton-containing fabrics (as compared to the fabric priorto treatment) when these components are incorporated into a textiletreatment medium and the fabric is treated with this medium. In additionto the above, EG III possesses substantial activity at alkaline pHswhere many detergent compositions are employed.

"Exo-cellobiohydrolase ("CBH") components" refer to the CBH componentsof Trichoderma spp. including the CBH I and CBH II components ofTrichoderma reesei. When used in the absence of the EG components ofTrichoderma spp, the CBH components of Trichoderma spp. alone do notimpart significant color retention/restoration and improved feel to theso-treated cotton-containing fabrics. Additionally, when used incombination with such EG components, the CBH I component of Trichodermareesei can impart enhanced strength loss and incremental cleaningbenefits to cotton-containing fabrics.

"β-Glucosidase (BG) components" refer to those components of cellulasewhich exhibit BG activity; that is to say that such components will actfrom the non-reducing end of cellobiose and other solublecellooligosaccharides ("cellobiose") and give glucose as the soleproduct. BG components do not adsorb onto or react with cellulosepolymers. Furthermore, such BG components are competitively inhibited byglucose (K_(i) approximately 1 mM). While in a strict sense, BGcomponents are not literally cellulases because they cannot degradecellulose, such BG components are included within the definition of thecellulase system because these enzymes facilitate the overalldegradation of cellulose by further degrading the inhibitory cellulosedegradation products (particularly cellobiose) produced by the combinedaction of CBH components and EG components. Without the presence of BGcomponents, moderate or little hydrolysis of crystalline cellulose willoccur. BG components are often characterized on aryl substrates such asp-nitrophenol B-D-glucoside (PNPG) and thus are often calledaryl-glucosidases. It should be noted that not all aryl-glucosidases areBG components, in that some do not hydrolyze cellobiose.

2. Methodology

The present invention is directed in part, to the discovery that anaqueous solution containing EG III, and substantially free of othercellulase proteins can be obtained from an aqueous enzyme mixture by theaddition of a low molecular weight alcohol. Surprisingly, under theseconditions, substantially all of the cellulase proteins, other than EGIII precipitate from solution leaving the solution containing EG IIIsubstantially free of other cellulase proteins. It has also been foundthat, under these conditions, substantially all of the cellulaseproteins, except EG III precipitate leaving a solution enriched inxylanase.

In one preferred method of carrying out the present invention, anaqueous mixture containing cellulase, was filtered to remove cell debrisand other solids and produced a liquid filtrate containing a mixture ofenzymes including cellulase proteins. More preferably, a cell freecellulase composition, such as CYTOLASE 123 (commercially available fromGenencor International, Inc., South San Francisco, Calif.) is used. Inanother method, the aqueous mixture could be obtained from any aqueoussource including aqueous mixtures already enriched for EG III. Moreparticularly the resulting EG III solution described in concurrentlyfiled application, U.S. Ser. No. 862,846, entitled METHODS FOR PRODUCINGSUBSTANTIALLY PURE EG III USING POLYETHYLENE GLYCOL, which isincorporated herein in its entirety by reference.

After the filtrate is obtained from the filtration step, organic saltsmay be added to the filtrate before contacting the filtrate with thealcohol. Without being limited to any theory, it is believed that insome cases, the addition of an organic salt may enhance the retention ofthe EG III component in solution.

Prior to addition of the alcohol, the pH of the aqueous mixture ispreferably adjusted to at least about pH=7, more preferably a pH ofabout 7 to about 12, and even more preferably a pH of about 7.5 to about10.5, most preferably to a pH of about 9.5.

Upon addition of an effective amount of low molecular weight alcohol tothe aqueous mixture, the enzymes other than the EG III component areprecipitated out of solution and the EG III component is preferentiallyretained in solution. The low molecular weight alcohol is added to theaqueous mixture under conditions which result in the precipitation ofcellulase proteins other than EG III with the retention of EG III in theaqueous solution. The specific time and temperature constraints employedin this step are not critical but depend on the degree of purity and theamount of recovery desired. For example, longer holding time will leadto higher degrees of purity as will lower temperatures. The specificcombination of time and temperature employed is within the skill of theart. In a preferred embodiment, such conditions would include atemperature range from about 10° C. to 30°C., more preferably from about15° C. to 25°C.

The low molecular weight alcohol is mixed with the aqueous mixture in apreferred embodiment for about 1 minute to 5 hours, more preferably from1 minute to 1 hour. The aqueous solution is then centrifuged andfiltered to remove the precipitated contaminating enzymes resulting in afirst supernate. The amount of EG III comprises at least approximately50% of the total cellulase protein in the first supernate as determinedby the gel electrophoresis method of Example 7.

The EG III component can then be purified from the first supernate by avariety of methods. In a preferred embodiment, an equal volume of coldethanol is then added to the first supernate and the precipitatecontaining EG III is collected by centrifugation. Alternatively, the EGIII component can be purified by ionic exchange chromatography (e.g., bymethods described in the examples hereinbelow). The EG III component isthen resuspended in a suitable buffer. Suitable buffers may be 10 mMsodium acetate pH=4.5, 10 mM sodium citrate pH=4.0 or other compatiblebuffers known in the art (i.e., buffers which do not denature the EG IIIcellulase component).

In another preferred method of carrying out the process of the presentinvention, a compatible inorganic salt is added to the first supernate.The addition of a sufficient amount of a compatible inorganic salt tothe first supernate results in the precipitation of substantially allcellulase proteins and xylanase remaining in solution, other than the EGIII component. This mixture is then centrifuged and filtered to removethe precipitated contaminating proteins resulting in a second supernate.EG III comprises at least 80% of the total cellulase protein in thesecond supernate, as determined by gel electrophoresis. The EG III canbe further purified from the second supernate by a variety of methods asdisclosed above.

One of the essential features of the process however, is the use of alow molecular weight alcohol. The alcohol has been found to be uniquelyactive and selective for precipitating cellulase proteins other than theEG III component, from aqueous mixtures containing numerous othercellulase components. The term "low molecular weight alcohol" as used inthis invention means a C₁ to C₃ alcohol [eg. ethanol, methanol, propanoland reagent alcohol (about 95% ethanol and about 5% methanol)] or amixture of the same. An "effective amount of a low molecular weightalcohol" is that amount added to the aqueous mixture which is necessaryto selectively precipitate a sufficient amount of the cellulaseproteins, except the EG III component, from the aqueous mixture toprovide a substantially pure EG III component once the precipitatedproteins are removed. Preferably the amount of a low molecular weightalcohol added is from about 1.5 to about 2.5 parts (v/v) of alcohol pervolume of aqueous mixture, more preferably the amount is 2.0 to about2.4 parts (v/v), most preferably the amount is about 2.2 parts (v/v).

It has been found that the amount of alcohol added involves a trade offbetween recovery and purity. At 1.5 parts alcohol, there is a greaterrecovery of EG III component, but more contamination by other proteins.At 2.5 parts alcohol, the EG III component is contaminated with onlyxylanase, but the recovery is lower. The best separation and recoveryhas been found at 2.2 parts (v/v) of alcohol.

The low molecular weight alcohol has been found to be particularlyuseful in separating the EG III cellulase component from an aqueousmixture of cellulase proteins because the aqueous mixture contains ahigh percentage of other cellulase proteins relative to the percentageof EG III. For example, the normal distribution of cellulase componentsin the CYTOLASE 123 cellulase system is believed to be as follows:

    ______________________________________                                        CBH I             45-55 weight percent                                        CBH II            13-15 weight percent                                        EG I              11-13 weight percent                                        EG II             8-10 weight percent                                         EG III            1-4 weight percent                                          BG                0.5-1 weight percent                                        ______________________________________                                    

Useful quantities of EG III component are obtained by the method of thisinvention. The loss of recovery of EG III component by the method ofthis invention, as compared to other methods, is compensated for by thespeed of recovery of the EG III component. The procedure does notrequire extensive fractionation steps for purification, although suchsteps can be followed for further purification if desired. Further, thecost of the starting material is negligible as compared to the highvalue of the purified EG III component.

The term "organic salt" as it is used in this application means anorganic salt containing at least one carbon atom and preferably 1 to 7carbon atoms which when used in conjunction with the low molecularweight alcohol facilitates the purification of EG III. Such organicsalts include, by way of example, sodium acetate, zinc acetate, sodiumformate and sodium benzoate and the like. The concentration of theorganic salt in the aqueous mixture can be varied to provide the desiredresult. Preferably, the amount of organic salt used is less than about30% (w/v) per original volume of aqueous mixture, more preferably theamount is between about 5% to 20% (w/v), most preferably the amount is10% (w/v). It is possible that the addition of an organic salt to theaqueous mixture prior to the addition of the low molecular weightalcohol may not be necessary where organic salts, such as sodiumbenzoate, are already present in the aqueous mixture. However, withoutthe addition of the organic salt, the amount of the EG III componentrecovered would be reduced.

The term "inorganic salt" means a compatible inorganic salt which whenused in conjunction with a low molecular weight alcohol facilitates thepurification of EG III without denaturing the enzyme. Suitable inorganicsalts include salts having a sulfate or ammonium ion, more preferably,ammonium sulfate. An "effective amount of a inorganic salt" is thatamount which when added to an aqueous mixture containing alcohol willresult in the precipitation of proteins other than the EG III componentfrom the solution to provide a substantially pure EG III component afterremoval of the precipitate. Preferably, the amount of a inorganic saltadded is an amount which creates a saturated solution.

The EG III cellulase component can be purified from the first filteredsupernate or the second filtered supernate by methods known in the art.For example, the addition of an equal volume of cold ethanol to thesupernate causes the precipitation of the Eg III component. Theprecipitate is collected and resuspended in an appropriate buffer.Suitable buffers are known in the art, for example 10 mM sodium acetatepH 4.5 and 10 mM sodium citrate pH 4.0. Alternatively, the EG IIIcomponent can be purified by ionic exchange chromatography methods knownin the art.

In another embodiment, the inorganic salt may be added to the originalaqueous mixture containing cellulase. The addition of a low molecularweight alcohol to this aqueous mixture will result in the precipitationof enzymes, other than EG III. This mixture is then centrifuged andfiltered to remove the precipitated contaminating enzymes. The EG IIIcomponent can be purified from the supernate by a variety of methods asdisclosed above.

In another preferred embodiment of carrying out the process of thepresent invention, the EG III component obtained from either of thethree methods described above, removed from the supernate andresuspended in an appropriate buffer can be further purified byfractionation. The solution will be desalted using a Sephadex G-25 gelfiltration resin column with 10 mM sodium phosphate buffer at pH 6.8.The desalted solution, would then be loaded onto a QA Trisacryl M anionexchange resin column. The fraction not bound on this column wouldcontain EG III. This fraction will be desalted using a Sephadex G-25 gelfiltration resin column equilibrated with 10 mM sodium citrate, pH 4.5.This solution will be again loaded onto a SP Trisacryl M cation exchangeresin column and the EG III cellulase component eluted with an aqueoussolution of 200 mM sodium chloride. The above process is described inconcurrently filed application U.S. Ser. No. 862,846, entitled METHODSFOR PRODUCING SUBSTANTIALLY PURE EG III CELLULASE USING POLYETHYLENEGLYCOL, which is incorporated herein in its entirety.

In another preferred method of carrying out the process of the presentinvention, the EG III sample obtained from the cation exchange columncan be further fractionated. The EG III sample will be desalted with aSephadex G-25 column which had been previously equilibrated with 10 mMsodium citrate pH 4. The solution is then applied to a FPLC system usinga Mono-S-HR 5/5 column (available from Pharmacia LKB Biotechnology,Piscataway, N.J.). The column will be eluted with 0-200 mM aqueousgradient of sodium chloride at a rate of 0.5 ml/minute.

It will be recognized that the above descriptions are preferred methodsof carrying out the process of the present invention and that numerousvariations of the above methods can be made in the process following theteachings of this invention. The various process conditions can bealtered and reagents used can be changed to provide various desired oroptimum operating conditions for recovery of the EG III cellulasecomponent from any suitable aqueous mixture of enzymes containing the EGIII component.

As will be recognized by those skilled in the art, the acids, bases andsalts referred to above in the description of the process of thisinvention can be changed or substituted with equivalent acids, bases orsalts which provide the desired pH or the desired salt content withoutinterfering with the operation of the invention and which do notdenature the EG III cellulase component.

EG III cellulase can be purified from any strain of Trichoderma spp.which produces EG III under suitable fermentation conditions or from anyother microorganism producing cellulase proteins including EG III. Whilethe particular source of EG III is not critical, preferred sources areTrichoderma reesei and Trichoderma viride. A particularly preferredsource of EG III from Trichoderma reesei is CYTOLASE 123 cellulase whichis commercially available from Genencor International, Inc., 180 KimballWay, South San Francisco, Calif. 94080.

In order to enhance the efficiency of the isolation of EG III, it may bedesirable to employ Trichoderma reesei genetically modified so as tooverexpress EG III and/or to be incapable of producing one or more of EGI, EG II, CBH I and/or CBH II components or xylanase. This willnecessarily lead to more efficient isolation of the EG III component by,for example, the alcohol extraction as described above. For example,substantially pure EG III prepared by fractionation methods set forth inthe Examples below was employed to determine the amino acid sequence ofparts of the protein using known sequencing methods (Example 4). Thisinformation can be used to prepare synthetic DNA probes in order toclone the gene encoding the EG III cellulase component. Once the EG IIIgene is cloned, it could be manipulated by recognized techniques andultimately inserted into various Trichoderma spp. strains or into othermicroorganisms. See, for example, U.S. Ser. No. 07/770,049 filed Oct. 4,1991, a continuation-in-part of U.S. Ser. No. 07/593,919, filed Oct. 5,1990 and U.S. Ser. No. 07/668,640, filed Mar. 13, 1991, all of whichdisclose methods for genetically engineering Trichoderma reesei so thatthe modified microorganism is incapable of expressing one or more of thecellulase genes or xylanase genes and, in fact, may overproduce anothercellulase gene. The disclosures of U.S. Ser. No. 07/770,049, filed Oct.4, 1991, U.S. Ser. No. 07/593,919, filed Oct. 5, 1990 and U.S. Ser. No.07/668,640, filed Mar. 13, 1991, are incorporated herein by reference intheir entirety.

Using the methods described in these applications, Trichoderma reeseican be genetically manipulated so as to produce EG III with or withoutother cellulase proteins. Moreover, the methods described in thoseapplications create Trichoderma reesei strains which do not produce anyheterologous proteins.

Additionally, it would be possible to express the EG III-encoding genein other microorganisms, including, but not limited to, yeast speciessuch as Sacchromyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Yarrowia lipolytica, Schanniomyces occidentals,etc. See, for example, PCT application Publication No. WO 85/04672. Inorder to obtain expression in these alternative, non-Trichoderma hosts,it may be necessary to functionally combine the EG III-coding DNAsequence with promoter and terminator sequences obtained from a genefrom that particular host. It may also be necessary to substitute theDNA sequence encoding a secretion signal sequence from the alternativehost for the DNA sequence encoding the EG III secretion signal sequence.Production and secretion of EG III in other organisms could enable EGIII to be obtained in substantially pure form.

The substantially pure EG III cellulase described above can be furtherprocessed into a liquid diluent, granules, emulsions, gels, pastes, orthe like. Such forms are well known to the skilled artisan. When a soliddetergent composition is desired, the cellulase composition ispreferably formulated as granules. Preferably, the granules can beformulated so as to contain a cellulase protecting agent. See, forinstance, U.S. Ser. No. 07/642,669 filed Jan. 17, 1991 as AttorneyDocket No. 010055-073 and entitled "GRANULES CONTAINING BOTH AN ENZYMEAND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONS CONTAININGSUCH GRANULES" which application is incorporated herein by reference inits entirety. Likewise, the granules can be formulated so as to containmaterials to reduce the rate of dissolution of the granule into the washmedium. Such materials and granules are disclosed in U.S. Ser. No.07/642,596 filed on Jan. 17, 1991 as Attorney Docket No. GCS-171-US1 andentitled "GRANULAR COMPOSITIONS" which application is incorporatedherein by reference in its entirety.

The following examples are offered to illustrate the present inventionand should not be construed in any way as limiting the scope of thisinvention.

EXAMPLES Example 1 Large Scale Purification of EG III From Cytolase 123cellulase A. Organic Salt and Low Molecular Weight Alcohol

To a cell free cellulase filtrate, CYTOLASE 123, (commercially availablefrom Genencor International, Inc., South San Francisco, Calif., which isproduced from wild type Trichoderma reesei), was added 10% sodiumacetate (w/v). The pH was adjusted to 9.5 by the addition of 50% NaOH.After the acetate was dissolved, 2.2 parts (v/v) ethanol at roomtemperature were added with mixing to 1 part of the cellulase filtrate(volume based on the starting volume of the filtrate). The ethanolfiltrate mixture was centrifuged at 10,000× g for 10 minutes and theprimary supernate was collected and filtered. The primary supernatecontains the EG III component. To this filtered supernate was added anequal volume of ethanol at -15° C. This mixture was centrifuged at10,000× g for 10 minutes and the precipitate collected. This precipitatewas resuspended in buffer.

It has been determined by RBB-CMC activity by the method as described inExample 6 that approximately 21% to 100% of the total amount of EG IIIis recovered from the cellulase filtrate by this method. It wasdetermined by gel electrophoresis, as described in Example 7, that theEG III component comprises at least approximately 50% of the totalcellulase protein in the precipitate. The cellulase filtrate of thisexample further contains xylanase whose concentration has been enrichedby this process (i.e., an increase in xylanase concentration by at least4 fold) and another unknown contaminating protein.

The pH does not have to be adjusted after the addition of sodiumacetate, but increasing the pH to about 9.5 improves the purification.

Other alcohols have been tried in place of ethanol. Methanol, propanoland reagent alcohol (95% ethanol and 5% methanol) give very similareffects. Reagent alcohol is as useful as pure ethanol and is lessexpensive.

Other salts have been tried in place of sodium acetate: zinc acetate,sodium formate and sodium benzoate. The zinc acetate appears to beequally good as the sodium salt. The formate and benzoate gave slightlyelevated, though still acceptable, levels of contaminating proteins.

B. Addition of an Inorganic Salt

In another experiment, sufficient ammonium sulfate to result in anexcess of a saturated solution was added to the primary filteredsupernate of the above method at room temperature and mixed forapproximately 1 hour. This mixture was centrifuged at 10,000× g for 10minutes and the resultant secondary supernate collected and filtered.The secondary supernate contains the EG III component. The precipitatecontains enriched xylanase. An equal volume of ethanol at -15° C. wasadded to this secondary supernate and mixed for approximately 5 minutes,and the precipitate was collected by centrifugation at 10,000×g for 10minutes. The precipitate was resuspended in buffer. 15 The addition ofthe ammonium sulfate reduces the amount of EG III component recovered ofthe total EG III component present in the original cellulase filtratebut increases the level of purity of the Eg III component. The EG IIIcomponent comprises at least 80% of the total cellulase protein in theprecipitate, as determined by gel electrophoresis.

Likewise, EG III cellulase from other strains of Trichoderma spp. can bepurified. For example, EG III cellulase derived from Trichoderma viridehas been described by Voragen et al., Methods in Enzymology,160:243-249. This reference describes the EG III cellulase as having amolecular weight of about 23.5 Kdaltons, a pH optimum of 5.5, and a pIof 7.7.

In order to enhance the efficiency of the isolation of EG III it may bedesirable to employ Trichoderma reesei genetically modified so as tooverexpress EG III and/or to be incapable of producing one or more EG I,EG II, CBHI and/or CBH II Components or xylanase components.

Example 2 Purification of EG III Via Fractionation

The substantially pure EG III component from the first supernate orsecond supernate obtained by in part a) of this example may be furtherpurified by fractionation after precipitation and resuspension in anappropriate buffer. Additionally, the original cellulase filtrate can bepurified by this method. Specifically, the fractionation is done usingcolumns containing the following resins: Sephadex G-25 gel filtrationresin from Sigma Chemical Company (St. Louis, Mo.), QA Trisacryl M anionexchange resin and SP Trisacryl M cation exchange resin from IBFBiotechnics (Savage, Md.).

In this example, CYTOLASE 123 cellulase, 0.5g, was desalted using acolumn of 3 liters of Sephadex G-25 gel filtration resin with 10 mMsodium phosphate buffer at pH 6.8. The desalted solution, was thenloaded onto a column of 20 ml of QA Trisacryl M anion exchange resinequilibrated with 10 mM sodium phosphate buffer pH=6.8. The fractionbound on this column contained CBH I and EG I. The fraction not bound onthis column contains CBH II, EG II and EG III. These fractions weredesalted using a column of Sephadex G-25 gel filtration resinequilibrated with 10 mM sodium citrate, pH 4.5. This solution, 200 ml,was then loaded onto a column of 20 ml of SP Trisacryl M cation exchangeresin. The EG III was eluted with 100 mL of an aqueous solution of 200mM sodium chloride.

One particular method for further purifying EG III is by furtherfractionation of an EG III sample obtained in this Example 2. Thefurther fraction was done on a FPLC system using a Mono-S-HR 5/5 column(available from Pharmacia LKB Biotechnology, Piscataway, N.J.). The FPLCsystem consists of a liquid chromatography controller, 2 pumps, a dualpath monitor, a fraction collector and a chart recorder (all of whichare available from Pharmacia LKB Biotechnology, Piscataway, N.J.). Thefractionation was conducted by desalting 5 ml of the EG III sampleprepared in this Example 2 with a 20 ml Sephadex G-25 column which hadbeen previously equilibrated with 10 mM sodium citrate pH 4. Thesolution was loaded onto the mono-S-HR 5/5 column previouslyequilibrated with 10 mM sodium citrate pH=4.0 and then eluted with 0-200mM aqueous gradient of NaCl at a rate of 0.5 ml/minute with samplescollected in 1 ml fractions. EG III was recovered in fractions 10 and 11and was determined to be greater than 90% pure by gel electrophoresis.EG III of this purity is suitable for determining the N-terminal aminoacid sequence by known techniques.

Substantially pure EG III has the following characteristics which arecompared to the other endoglucanases isolated from Trichoderma reesei.

                  TABLE I                                                         ______________________________________                                                 MW        pI       pH optimum.sup.1                                  ______________________________________                                        EG I       ˜47-49 kD                                                                           4.7      ˜5                                      EG II      ˜35 kD                                                                              5.5      ˜5                                      EG III     ˜25-28 kD                                                                           7.4      ˜5.5-6.0                                ______________________________________                                         1. pH optimum determined by RBBCMC activity as per Example 3 below.      

As can be seen from the above table, EG III has both a higher pH optimumand a higher pI as compared to the other endoglucanase components ofTrichoderma reesei. In Example 3 below, it is seen that EG III alsoretains significant RBB-CMC activity under alkaline pHs.

Likewise, EG III cellulase from other strains of Trichoderma spp. can bepurified. For example, EG III cellulase derived from Trichoderma viridehas been described by Voragen et al., Methods in Enzymology,160:243-249. This reference describes the EG III cellulase as having amolecular weight of about 23.5 Kdaltons, a pH optimum of 5.5, and a pIof 7.7.

In order to enhance the efficiency of the isolation of EG III it may bedesirable to employ Trichoderma reesei genetically modified so as tooverexpress EG III and/or to be incapable of producing one or more EG I,EG II, CBHI and/or CBH II components or xylanase components.

Example 3 Activity of Cellulase Compositions Over a pH Range

The following procedure was employed to determine the pH profiles of twodifferent cellulase compositions. The first cellulase composition was aCBH I and II deleted cellulase composition prepared from Trichodermareesei genetically modified in a manner similar to that described belowso as to be unable to produce CBH I and CBH II components. Insofar asthis cellulase composition does not contain CBH I and CBH II whichgenerally comprise from about 58 to 70 percent of a cellulasecomposition derived from Trichoderma reesei, this cellulase compositionis necessarily enriched in EG components. Since EG III is the most minorof the endoglucanase components of Trichoderma reesei , this compositionpredominates in EG I and EG II components.

The second cellulase composition was an approximately 20-40% purefraction of EG III isolated from a cellulase composition derived fromTrichoderma reesei via purification methods similar to Example 2. Theactivity of these cellulase compositions was determined at 40° C. andthe determinations were made using the following procedures.

Add 5 to 20 μl of an appropriate enzyme solution at a concentrationsufficient to provide the requisite amount of enzyme in the finalsolution. Add 250 μl of 2 weight percent RBB-CMC (Remazol Brilliant BlueR-Carboxymethyl-cellulose--commercially available from MegaZyme, 6Altona Place, North Rocks, N.S.W. 2151, Australia) in 0.05Mcitrate/phosphate buffer at pH 4, 5, 5.5, 6, 6.5, 7, 7.5 and 8.

Vortex and incubate at 40° C. for 30 minutes. Chill in an ice bath for 5to 10 minutes. Add 1000 μl of methyl cellosolve containing 0.3M sodiumacetate and 0.02M zinc acetate. Vortex and let sit for 5-10 minutes.Centrifuge and pour supernatant into cuvettes.

Relative enzyme activity was determined by measuring the optical density(OD) of the solution in each cuvette at 590 nm. Higher levels of opticaldensity correspond to higher levels of enzyme activity.

The results of this analysis are set forth in FIG. 1 which illustratesthe relative activity of the CBH I and II deleted cellulase compositioncompared to the EG III cellulase composition. From this figure, it isseen that the cellulase composition deleted in CBH I and CBH IIpossesses optimum cellulolytic activity against RBB-CMC at near pH 5.5and has some activity at alkaline pHs, i.e., at pHs from above 7 to 8.On the other hand, the cellulase composition enriched in EG IIIpossesses optimum cellulolytic activity at about pH 5.5-6 and possessessignificant activity at alkaline pHs.

Example 4 Isoelectric Focusing Gels

The purpose of this example is to illustrate isoelectric focusing gelsof different EG III cellulase compositions. Specifically, cellulaseproduced by a wild type Trichoderma reesei; cellulase derived from astrain of Trichoderma reesei transformed so as to be incapable ofexpressing EG I and EG II cellulase proteins; and substantially pure EGIII cellulase via purification methods similar to Example 2 wereanalyzed on isoelectric focusing gels.

Samples of these cellulases were analyzed by isoelectric focusing gelsusing a Pharmacia IEF system (FBE-3000, Pharmacia Inc., Piscataway,N.J.) and pH 3-10 precast gels (Servalyt Precote, available from Serva,Carl-Berg, Germany) according to the manufacturer's instructions. Thegels were stained with Ephortec™ stain (Serva Blue W, available fromSerra Fine Biochemicals, Westbury, N.Y. 11590) to visualize the proteinbands. The resulting gel is set forth in FIG. 2; wherein Lane 1 of FIG.2 illustrates the isoelectric focusing gel of cellulase derived from awild strain of Trichoderma reesei; Lane 2 illustrates the isoelectricfocusing gel of cellulase derived from a strain of Trichoderma reesei soas to be incapable of expressing EG I and II; and Lane 3 illustrates theisoelectric focusing gel of substantially pure EG III cellulase obtainedusing the method of Example 2. In this figure, the margin adjacent toLane I is marked to identify the bands corresponding to cellulaseproteins so as to permit identification of the proteins.

This figure demonstrates that EG III is not a degradation product ofeither EG I or EG II proteins because, in Lane 2 of this figure, theseproteins are not present while the EG III protein

Example 5 Peptide Sequencing of EG III

The substantially pure EG III component obtained by the purificationmethod of Example 2, was precipitated by the addition of 0.9 ml ofacetone to 0.1 ml of protein solution (at a concentration of 1 mg/ml)and incubation at -20° C. for 10 minutes. The protein was collected bycentrifugation and the pellet dried and resuspended in 0.01 ml of 8Murea in 88% formic acid and 0.01 ml of cyanogen bromide (200 mg/ml) in88% formic acid. The mixture was incubated at room temperature for fourhours.

Individual peptides were purified on a HPLC (high pressure liquidchromatography) column. A Synchropak RP-4 column was equilibrated indeionized milliQ water with 0.05% TEA (triethylamine) and 0.05% TFA(trifluoroacetic acid). The sample was loaded onto the HPLC column andelution was carried out with 100% acetonitrile and 0.05% TEA and 0.05%TFA, with a gradient of 1% per minute. The amino-terminal regions ofisolated peptides were sequenced by the method of Edman using a fullyautomated apparatus. The amino acid sequence obtained from two fragmentsof the EG III component are shown in FIG. 3

Example 6 Determination of Recovery of EG III Cellulase

20 μL of sample (or standard) was added to an individual eppendorf tube.Using an Eppendorf Repeat Pipette™, 250 μl of substrate is added tosamples and standards. All the eppendorf tubes were immediatelyvortexed. The tubes were then incubated in a 37° C. water bath for 30minutes. At the end of incubation, 1 ml of precipitant was added to eachtube using an Eppendorf Repeat Pipette. The tubes were vortexedvigorously. The samples were centrifuged for 3 minutes at 5,000×g. Thesupernate was poured into disposable cuvettes and the OD at 590 nm wasdetermined using the OU/ml standard as blank.

The samples were diluted such that their activity fell within thestandard range of 1.5 to 6.0 Units/mi. Samples were run in duplicate.

The standard was Genencor CYTOLASE 123 lot 87111. This is defined ascontaining 1000 RBB-CMC Units/ml. Appropriate dilutions were made tomake standard solutions containing 0, 1.5, 3.0, 4.5 and 6.0 Units/ml.Standards were run in duplicate.

The substrate was prepared by adding 2 gms dry RBB-CMC(Azo-CM-Cellulose, obtained from MegaZyme, Ltd.) to 80 ml Just boileddeionized water. The mixture is stirred vigorously as it cooled to roomtemperature until all of the substrate had solubilized. 5.0 ml of a 2Msodium acetate solution was added. The pH of the solution was adjustedto 4.5 and the volume to 100 ml. A 1/100 dilution of a 2% solution ofsodium azide was added to yield a final concentration of 200 ppm.

The precipitate was prepared by adding 33 grams anhydrous sodium acetateand. 4 grams zinc acetate to 150 ml distilled water. The pH of thesolution was adjusted to 5.0 with 5M HCl and the volume was adjusted to200 ml and 800 ml of ethanol was added.

The following results and estimates were obtained:

                  TABLE II                                                        ______________________________________                                                   RBB-CMC                                                                       UNITS                                                                         ATTRIBUTABLE   PERCENTAGE                                          SAMPLE     TO EG III      ESTIMATED                                           ______________________________________                                        aqueous mixture                                                                          2025-10800     100%                                                first supernate                                                                          2300           21-100%                                             from Example 1                                                                ______________________________________                                    

EG III is believed to have a specific activity of 15 to 20 RBB units permg of protein.

Example 7 SDS-PAGE Gels of EG III Component

Samples to be run are diluted to contain approximately 1 to 3 mg ofprotein per ml. 100 μl of sample is placed in an eppendorf tube with 25μl of 5X PDS, vortexed and heated at 98° C. for 5 minutes. Next, 12 μlof each sample is removed and loaded into a well. The gel is run at 40mA with constant current for approximately 90 minutes in running bufferdiluted to 1× strength with distilled water.

At completion the gel is removed from between the glass plates andimmersed in a solution of Destain for 30 minutes with mild agitation tofix the protein. The gel is next stained with Coomasie Blue Stain withglacial acetic acid for 1 hour with mild agitation. The background stainis removed in Destain solution for approximately 18 hours.

Prepoured gels were obtained from Daiichi Pure Chemicals Co., Ltd. A gelwith a gradient of 10 to 20% acrylamide or a non-gradient of 12.5%acrylamide was used. The electrophoresis was carried out in a Daiichielectrophoresis box.

5XPDS contains 2.5 ml of 20% sodium dodecylsulfate; 1 ml glycerol; 0.5ml of 0.5M sodium phosphate pH 6.6; 1 ml distilled water; 0.1 mlbeta-mercaptoethanol; and 10 mg bromophenol blue. The running buffercontains 30.25 g Tris base; 144.5 g ultra pure glycine; milli-Q H₂ O to1 liter; 5 mls 20% SDS. The Destain contains 82.5 ml glacial aceticacid, 200 ml ethanol and dH₂ to 1 liter. The Coomasie Blue Staincontains brilliant blue (Sigma No. B-0630) 2.5 g; ethanol 250 ml; dH₂ Oto 1 liter. Before use mix 90 ml of coomasie blue stain with 10 mlglacial acetic acid.

The results are indicated in FIG. 4 which illustrates in Lane 11 theproteins expressed by a strain of Trichoderma reesei transformed so asto be incapable of expressing EG I and EG II components; and in lane 2the proteins found in substantially pure EG III cellulase obtained bythe method of Example 1 from a strain of T. reesei transformed so as tobe incapable of expressing EG I and EG II components in which 10% w/v ofzinc acetate and 2 parts ethanol were added to the cellulase mixture.

Example 8 Selection for Pyr4 Derivatives of Trichoderma reesei

The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, anenzyme required for the biosynthesis of uridine. The toxic inhibitor5-fluoroorotic acid (FOA) is incorporated into uridine by wild-typecells and thus poisons the cells. However, cells defective in the pyr4gene are resistant to this inhibitor but require uridine for growth. Itis, therefore, possible to select for pyr4 derivative strains using FOA.In practice, spores of T. reesei strain RL-P37 [Sheir-Neiss, G. andMontenecourt, B. S., Appl. Microbiol. Biotechnol. 20, p. 46-53 (1984)]were spread on the surface of a solidified medium containing 2 mg/mluridine and 1.2 mg/ml FOA. Spontaneous FOA-resistant colonies appearedwithin three to four days and it was possible to subsequently identifythose FOA-resistant derivatives which required uridine for growth. Inorder to identify those derivatives which specifically had a defectivepyr4 gene, protoplasts were generated and transformed with a plasmidcontaining a wild-type pyr4 gene (see Examples 10 and 11). Followingtransformation, protoplasts were plated on medium lacking uridine.Subsequent growth of transformed colonies demonstrated complementationof a defective pyr4 gene by the plasmid-borne pyr4 gene. In this way,strain GC69 was identified as a pyr4 derivative of strain RL-P37.

Example 9 Preparation of CBHI Deletion Vector

A cbh1 gene encoding the CBHI protein was cloned from the genomic DNA ofT. reesei strain RL-P37 by hybridization with an oligonucleotide probedesigned on the basis of the published sequence for this gene usingknown probe synthesis methods (Shoemaker et al., 1983b). The cbh1 generesides on a 6.5 kb PstI fragment and was inserted into PstI cut pUC4K(purchased from Pharmacia Inc., Piscataway, N.J.) replacing the Kan^(r)gene of this vector using techniques known in the art, which techniquesare set forth in Maniatis et al., (1989) and incorporated herein byreference. The resulting plasmid, pUC4K::cbh1 was then cut with HindIIIand the larger fragment of about 6 kb was isolated and relegated to givepUC4K::cbh1ΔH/H (see FIG. 5). This procedure removes the entire cbh1coding sequence and approximately 1.2 kb upstream and 1.5 kb downstreamof flanking sequences. Approximately, 1 kb of flanking DNA from eitherend of the original PstI fragment remains.

The T. reesei pyr4 gene was cloned as a 6.5 kb HindIII fragment ofgenomic DNA in pUC18 to form pTpyr2 (Smith et al., 1991) following themethods of Maniatis et al., supra. The plasmid pUC4K::cbhIΔH/H was cutwith HindIII and the ends were dephosphorylated with calf intestinalalkaline phosphatase. This end dephosphorylated DNA was ligated with the6.5 kb HindIII fragment containing the T. reesei pyr4 gene to givepΔCBHIpyr4. FIG. 5 illustrates the construction of this plasmid.

Example 10 Isolation of Protoplasts

Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract,2% glucose) in a 500 ml flask with about 5×10⁷ T. reesei GC69 spores(the pyr4 derivative strain). The flask was then incubated at 37° C.with shaking for about 16 hours. The mycelium was harvested bycentrifugation at 2,750× g. The harvested mycelium was further washed ina 1.2M sorbitol solution and resuspended in 40 ml of a solutioncontaining 5 mg/ml Novozym^(R) 234 solution (which is the trade name fora multi-component enzyme system containing 1,3-alpha-glucanase,1,3-beta-glucanase, laminarinase, xylanase, chitinase and protease fromNovo Biolabs, Danbury, Conn.); 5 mg/ml MgSO₄. 7H₂ O; 0.5 mg/ml bovineserum albumin; 1.2M sorbitol. The protoplasts were removed from thecellular debris by filtration through Miracloth (Calbiochem Corp, LaJolla, Calif.) and collected by centrifugation at 2,000 ×g. Theprotoplasts were washed three times in 1.2M sorbitol and once in 1.2Msorbitol, 50 mM CaCl₂, centrifuged and resuspended at a density ofapproximately 2×10⁸ protoplasts per ml of 1.2M sorbitol, 50 mM CaCl₂.

Example 11 Transformation of Fungal Protoplasts with pΔCBHIpyr4

200 μl of the protoplast suspension prepared in Example 10 was added to20 μl of EcoRI digested pΔCBHIpyr4 (prepared in Example 9) in TE buffer(10 mM Tris, pH 7.4; 1 mM EDTA) and 50 μl of a polyethylene glycol (PEG)solution containing 25% PEG 4000, 0.6M KCl and 50 mM CaCl₂. This mixturewas incubated on ice for 20 minutes. After this incubation period 2.0 mlof the above-identified PEG solution was added thereto, the solution wasfurther mixed and incubated at room temperature for 5 minutes. Afterthis second incubation, 4.0 ml of a solution containing 1.2M sorbitoland 50 mM CaCl₂ was added thereto and this solution was further mixed.The protoplast solution was then immediately added to molten aliquots ofVogel's Medium N (3 grams sodium citrate, 5 grams KH₂ PO₄, 2 grams NH₄NO₃, 0.2 grams MgSO₄.7H₂ O, 0.1 gram CaCl₂. 2H₂ O, 5 μg α-biotin, 5 mgcitric acid, 5 mg ZnSO₄. 7H₂ O, 1 mg Fe(NH₄)₂. 6H₂ O, 0.25 mg CuSO₄. 5H2O, 50 μg MnSO₄.4H20 per liter) containing an additional 1% glucose,1.2M sorbitol and 1% agarose. The protoplast/medium mixture was thenpoured onto a solid medium containing the same Vogel's medium as statedabove. No uridine was present in the medium and therefore onlytransformed colonies were able to grow as a result of complementation ofthe pyr4 mutation of strain GC69 by the wild type pyr4 gene insert inpΔCBHIpyr4. These colonies were subsequently transferred and purified ona solid Vogel's medium N containing as an additive, 1% glucose andstable transformants were chosen for further analysis.

At this stage stable transformants were distinguished from unstabletransformants by their faster growth rate and formation of circularcolonies with a smooth, rather than ragged outline on solid culturemedium lacking uridine. In some cases a further test of stability wasmade by growing the transformants on solid non-selective medium (i.e.containing uridine), harvesting spores from this medium and determiningthe percentage of these spores which will subsequently germinate andgrow on selective medium lacking uridine.

Example 12 Analysis of the Transformants

DNA was isolated from the transformants obtained in Example 8 after theywere grown in liquid Vogel's medium N containing 1% glucose. Thesetransformant DNA samples were further cut with a PstI restriction enzymeand subjected to agarose gel electrophoresis. The gel was then blottedonto a Nytran membrane filter and hybridized with a ³² P-labelledpΔCBHIpyr4 probe. The probe was selected to identify the native cbh1gene as a 6.5 kb PstI fragment, the native pyr4 gene and any DNAsequences derived from the transforming DNA fragment.

The radioactive bands from the hybridization were visualized byautoradiography. The autoradiograph is seen in FIG. 7. Five samples wererun as described above, hence samples A, B, C, D, and E. Lane E is theuntransformed strain GC69 and was used as a control in the presentanalysis. Lanes A-D represent transformants obtained by the methodsdescribed above. The numbers on the side of the autoradiograph representthe sizes of molecular weight markers. As can be seen from thisautoradiograph, lane D does not contain the 6.5 kb CBHI band, indicatingthat this gene has been totally deleted in the transformant byintegration of the DNA fragment at the cbh1 gene. The cbh1 deletedstrain is called P37PΔCBHI. FIG. 6 outlines the deletion of the T.reesei gene by integration through a double cross-over event of thelarger EcoRI fragment from pΔCBHIpyr4 at the cbh1 locus on one of the T.reesei chromosomes. The other transformants analyzed appear identical tothe untransformed control strain.

Example 13 Analysis of the Transformants with pIntCBHI

The same procedure was used in this example as in Example 12, exceptthat the probe used was changed to a ³² P labelled pIntCBHI probe. Thisprobe is a pUC-type plasmid containing a 2 kb BglII fragment from thecbh1 locus within the region that was deleted in pUC4K::cbh1ΔH/H. Twosamples were run in this example including a control, sample A, which isthe untransformed strain GC69 and the transformant P37PΔCBHI, sample B.As can be seen in FIG. 8, sample A contained the cbh1 gene, as indicatedby the band at 6.5 kb; however the transformant, sample B, does notcontain this 6.5 kb band and therefore does not contain the cbh1 geneand does not contain any sequences derived from the pUC plasmid.

Example 14 Protein Secretion by strain P37PΔCBHI.

Spores from the produced P37PΔCBHI strain were inoculated into 50 ml ofa Trichoderma basal medium containing 1% glucose, 0.14% (NH₄)₂ SO₄, 0.2%KH₂ PO₄, 0.03% MgSO₄, 0.03% urea, 0.75% bactotryptone, 0.05% Tween 80,0.000016% CuSO₄.5H2O, 0.001% FeSO₄.7H₂ O, 0.000128% ZnSO₄.7H₂ O,0.0000054% Na₂ MoO₄.2H₂ O, 0.0000007% MnCl.4H2O). The medium wasincubated with shaking in a 250 ml flask at 37° C. for about 48 hours.The resulting mycelium was collected by filtering through Miracloth(Calbiochem Corp.) and washed two or three times with 17 mM potassiumphosphate. The mycelium was finally suspended in 17 mM potassiumphosphate with 1 mM sophorose and further incubated for 24 hours at 30°C. with shaking. The supernatant was then collected from these culturesand the mycelium was discarded. Samples of the culture supernatant wereanalyzed by isoelectric focusing using a Pharmacia Phastgel system andpH 3-9 precast gels according to the manufacturer's instructions. Thegel was stained with silver stain to visualize the protein bands. Theband corresponding to the cbh1 protein was absent from the samplederived from the strain P37PΔCBHI, as shown in FIG. 9. This isoelectricfocusing gel shows various proteins in different supernatant cultures ofT. reesei. Lane A is partially purified CBHI; Lane B is the supernatantfrom an untransformed T. reesei culture; Lane C is the supernatant fromstrain P 37PΔCBHI produced according to the methods of the presentinvention. The position of various cellulase components are labelledCBHI, CBHII, EGI, EGII, and EGIII. Since CBHI constitutes 50% of thetotal extracellular protein, it is the major secreted protein and henceis the darkest band on the gel. This isoelectric focusing gel clearlyshows depletion of the CBHI protein in the P37PΔCBHI strain.

Example 15 Preparation of pPΔCBHII

The cbh2 gene of T. reesei, encoding the CBHII protein, has been clonedas a 4.1 kb EcoRI fragment of genomic DNA which is showndiagrammatically in FIG. 10A (Chen et al., 1987, Biotechnology,5:274-278). This 4.1 kb fragment was inserted between the EcoRI sites ofpUC4XL. The latter plasmid is a pUC derivative (constructed by R. M.Berka, Genencor International Inc.) which contains a multiple cloningsite with a symmetrical pattern of restriction endonuclease sitesarranged in the order shown here: EcoRI, BamHI, SacI, SmaI, HindIII,XhoI, BglII, ClaI, BqlII, XhoI, HindIII, SmaI, SacI, BamHI, EcoRI. Usingmethods known in the art, a plasmid, pPΔCBHII (FIG. 10B), has beenconstructed in which a 1.7 kb central region of this gene between aHindIII site (at 74 bp 3' of the CBHII translation initiation site) anda ClaI site (at 265 bp 3' of the last codon of CBHII) has been removedand replaced by a 1.6 kb HindIII-ClaI DNA fragment containing the T.reesei pyr4 gene.

The T. reesei pyr4 gene was excised from pTpyr2 (see Example 9) on a 1.6kb NheI-SphI fragment and inserted between the SphI and XbaI sites ofpUC219 to create p219M (Smith et al., 1991, Curr. Genet 19 p. 27-33).The pyr4 gene was then removed as a HindIII-ClaI fragment having sevenbp of DNA at one end and six bp of DNA at the other end derived from thepUC219 multiple cloning site and inserted into the HindIII and ClaIsites of the cbh2 gene to form the plasmid pPΔCBHII (see FIG. 10B).

Digestion of this plasmid with EcoRI will liberate a fragment having 0.7kb of flanking DNA from the cbh2 locus at one end, 1.7 kb of flankingDNA from the cbh2 locus at the other end and the T. reesei pyr4 gene inthe middle.

Example 16 Generation of a pyr4⁻ Derivative of P37PΔCBHI

Spores of the transformant (P37PΔCBHI) which was deleted for the cbh1gene were spread onto medium containing FOA. A pyr4⁻ derivative of thistransformant was subsequently obtained using the methods of Example 8.This pyr4⁻ strain was designated P37PΔCBHIPyr⁻ 26.

Example 17 Deletion of the cbh2 Gene in a Strain Previously Deleted forcbh1

Protoplasts of strain P37PΔCBHIPyr⁻ 26 were generated and transformedwith EcoRI digested pPΔCBHII according to the methods outlined inExamples 10 and 11.

Purified stable transformants were cultured in shaker flasks as inExample 14 and the protein in the culture supernatants was examined byisoelectric focusing, One transformant (designated P37PΔΔCBH67) wasidentified which did not produce any CBHII protein, Lane D of FIG. 9shows the supernatant from a transformant deleted for both the cbh1 andcbh2 genes produced according to the methods of the present invention.

DNA was extracted from strain P37PΔΔCBH67, digested with EcoRI andAsp718, and subjected to agarose gel electrophoresis. The DNA from thisgel was blotted to a membrane filter and hybridized with ³² P-labelledpPΔCBHII (FIG. 11). Lane A of FIG. 11 shows the hybridization patternobserved for DNA from an untransformed T. reesei strain. The 4.1 kbEcoRI fragment containing the wild-type cbh2 gene was observed. Lane Bshows the hybridization pattern observed for strain P37PΔCBH67. Thesingle 4.1 kb band has been eliminated and replaced by two bands ofapproximately 0.9 and 3.1 kb. This is the expected pattern if a singlecopy of the EcoRI fragment from pPΔCBHII had integrated precisely at thecbh2 locus.

The same DNA samples were also digested with EcoRI and Southern blotanalysis was performed as above. In this Example, the probe was ³² Plabelled pIntCBHII. This plasmid contains a portion of the cbh2 genecoding sequence from within that segment of the cbh2 gene which wasdeleted in plasmid pPΔCBHII. No hybridization was seen with DNA fromstrain P37PΔΔCBH67 showing that the cbh2 gene was deleted and that nosequences derived from the pUC plasmid were present in this strain.

Example 18 Construction of pΔEGIpyr-3 and Transformation of a pyr4Deficient Strain of T. reesei

The T. reesei egl1 gene, which encodes EGI has been cloned as a 4.2 kbHindIII fragment of genomic DNA from strain RL-P37 by hybridization witholigonucleotides synthesized according to the published sequence(Pentilla et al., 1986, Gene 45: 253-263; van Arsdell et al., 1987,Bio/Technology: 60-64).

This DNA fragment was inserted at the HindIII site of pUC100. Aninternal 1 kb EcoRV fragment which extended from a position close to themiddle of the EGI coding sequence to a position beyond the 3' end of thecoding sequence was removed by enzyme digestion and was replaced byligation with a 2.2 kb BamHI-HindIII fragment containing the cloned A.niger pyrG gene (Wilson et al., 1988, Nucl. Acids Res. 16 p. 2339) togive pΔEGIpyrG-3 (FIG. 12). Transformation of a pyr4 deficient strain ofT. reesei (strain GC69) by the method set forth in Examples 10 and 11,with pΔEGIpyr-3, after it had been digested with HindIII to release thefragment containing the pyrG gene with flanking regions from the egl1locus at either end, led to transformants in which the genomic egl1 genewas disrupted by a mechanism outlined in FIG. 13. DNA was extracted fromtransformants, digested with HindIII, subjected to agarose gelelectrophoresis and blotted onto a membrane filter. The filter washybridized with radiolabelled pΔEGIpyr-3. In the untransformed strain ofT. reesei the egl1 gene was present on a 4.2 kb HindIII fragment of DNA.However, following deletion of the egl1 gene by integration of thedesired fragment from pΔEGIpyr-3 this 4.2 kb HindIII fragmentdisappeared and was replaced by a HindIII fragment approximately 1.2 kblarger in size. This pattern was observed for one transformant, whichwas designated ΔEGI-3.

Example 19 Construction of the PAΔEGII-1 and Deletion of the EGII Gene

The egl3 gene, encoding EG II (previously also known as EG III), wascloned from T. reesei strain RL-P37 as a 4 kb PstI genomic DNA fragmentby hybridization with oligonucleotides synthesized according to thepublished sequence (Saloheimo et al., 1988, Gene 63:11-21). This DNAfragment was inserted into the PstI site of pUC18. This plasmid, pEGII,was subsequently digested with EcoRV to remove the entire EG II codingregion on an approximately 2 kb segment extending from a positionapproximately 180 bp 5' of the EGII coding region to a position a fewhundred base pairs beyond the end of the coding region. This segment wasreplaced with an SspI fragment of Aspergillus nidulans genomic DNAcontaining the amdS gene (Corrick et al., 1987, Gene 53:63-71) to createplasmid PAΔEGII-1 (See FIG. 14).

Wild-type strains of T. reesei are unable to grow on acetamide as a solenitrogen source. Transformation with the amdS gene confers this abilityand this is the basis for the selection system for transformantscontaining this gene.

Protoplasts of strain ΔEGI-3 were transformed, by the methods describedin Examples 10 and 11, with pAΔEGII-1 which had been digested withHindIII and EcoRI and transformants able to grow on acetamide wereselected. Subsequently, DNA was extracted from stable transformants,digested with pstI, subjected to agarose gel electrophoresis and blottedonto a membrane filter. The filter was hybridized with radiolabelledpAΔEGII-1. Homologous integration of the HindIII-EcoRI fragment frompAΔEGII-1, which contained egl3 flanking regions and amdS, at thegenomic egl3 locus in a transformant lead to the 4 kb genomic PstIfragment containing the egl3 gene being replaced by smaller Pst1fragments including two which would be approximately 1.0 and 2.8 kb inlength. This pattern of hybridization was observed for one transformantwhich was designated strain ΔΔEG-1. This strain has deletions in boththe EGI and EGII encoding genes and consequently is unable to produceeither of these proteins.

The methods described in Examples 8-19 and in U.S. Ser. No. 07/770,049,filed Oct. 4, 1991, (incorporated herein by reference in its entirety)may be used to obtain T. reesei transformants which are unable toproduce any or all of the following cellulase components; EG I, EG II,CBHI and CBHII, and the xylanase components. Additionally, the methodsdescribed may be used to obtain a T. reesei transformant whichoverexpresses the EG III cellulase component.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions omissions and changes may be made withoutdeparting from the spirit and scope thereof. Accordingly, it is intendedthat the scope of the present invention be limited solely by the scopeof the following claims, including equivalents thereof.

What is claimed is:
 1. A method for preparing an aqueous solutioncontaining both EG III and xylanase substantially free of othercellulase proteins from an aqueous mixture of cellulase proteinscontaining EG III, xylanase and other cellulase proteins whichcomprises:(a) adding an amount of a low molecular weight alcoholselected from the group consisting of ethanol, methanol, propanol andmixtures thereof and an organic salt to said aqueous mixture containingcellulase proteins under conditions wherein substantially all of thecellulase proteins other than EG III and xylanase are precipitated outof the aqueous mixture and (b) separating an aqueous supernatecontaining said EG III and xylanase from said aqueous mixture.
 2. Amethod according to claim 1 further comprising adding the organic saltto the aqueous mixture before the addition of the alcohol.
 3. A methodaccording to claim 2 wherein the organic salt comprises a salt selectedfrom the group consisting of sodium acetate, zinc acetate, sodiumformate and sodium benzoate.
 4. A method of claim 1 wherein the lowmolecular weight alcohol is ethanol.
 5. A method of claim 1 wherein thebeginning aqueous mixture is a filtered whole cell extract.
 6. A methodof claim 1 wherein the beginning aqueous mixture is a cell freecellulase mixture.
 7. A method of claim 1, further comprising adjustingthe pH of the aqueous mixture to a pH of at least about 7 before theaddition of the alcohol.
 8. A method of claim 7 wherein the pH of theaqueous mixture is adjusted to about 9.5.